The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin gcenes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline .delta.-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively. See Gaertner, F. H. (1989) "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255; see also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg et al (Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter(1983)Z. ang. Ent. 96:500-508) describe Bacillus thuringiensis var. tenebrionis, which is active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni.
Recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte, H., H. R. Whiteley (1989) Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were CryIII (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported. (Feitelson. J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). CryV has been proposed to designate a class of toxin genes that are nematode-specific.
Regular use of chemical control of unwanted organisms can select for chemical resistant strains. Chemical resistance occurs in many species of economically important insects and has also occurred in nematodes of sheep, goats, and horses. The development of chemical resistance necessitates a continuing search for new control agents having different modes of action.
Xenorhabdus spp. are a commercially significant group of pathogenic bacteria which can infect a variety of insect larvae via a nematode vector host. These bacteria exist in a symbiotic relationship with pathogenic nematodes of the genus Neoaplectana. Steinernema, and Heterorhabditis. This system provides some advantages for the biological control of insect pests because, unlike many other biocontrol agents (such as Bacillus thuringiensis), the nematode actively seeks out prey larvae.
Xenorhabdus spp. are Gram negative, facultatively anaerobic, rod shaped bacteria currently assigned to the family Enterobacteriaceae. Two species (X. nematophilus and X. luminescens) and four subspecies of X. nematophilus (nematophilus, bovienii, poinarii, and beddingii) have been described.
Xenorhabdus spp. are carried in the closed intestine of third instar, infective, juvenile nematodes such as Heterorhabditis. Heterorhabditis spp. possess a specialized, anterior tooth that allows those nematodes to scrape and rupture the hard, exterior cuticle of the insect in order to gain access to the haemocoel. Neoaplectana and Steinernema spp. possess high hydrostatic heads and have an extremely narrow diameter of their anterior region, which enables these nematodes to punch through the softer internal parts of the insect. Once entry to the haemocoel has been gained, these nematodes secrete toxin which inhibits the insect inducible immune system and release their insect-pathogenic bacterial symbiont, thus killing the host insect.
The degree of infectivity of each nematode species/strain for different hosts varies considerably. No one species/strain is most infective for a wide variety of insect hosts. In addition, each genus of nematode hosts a particular species of bacterium. In nematodes of the Heterorhabditis genus, the symbiotic bacterium is Photorhabdus luminescens. The interaction between the bacterium Xenorhabdus and the host nematode is also specific. Apparently, X. nematophilus are symbiotic with Steinernematidae and X. luminescens with Heterorhabditidea. X. nematophilus isolated from infective nematodes are able to be carried by only a small proportion of other nematodes. Reasons for this specificity are not completely understood, but this might be related to the production of bacterial cell surface components (e.g. fimbriae).
An insecticidal toxin from Photorhabdus luminescens that has activity only when injected into Lepidopteran and Coleopteran insect larvae is known. WO 97/17432 (Ensign et al.) reports that proteins from the genus Photorhabdus are orally toxic to insects upon exposure. As reported therein, Photorhabdus luminescens (formerly Xenorhabdus luminescens) were found in mammalian clinical samples and as a bacterial symbiont of entomopathogenic nematodes of the genus Heterorhabditis.
Although the foregoing was known in the art, the utility of nematodes as a source for other insecticidal bacteria (preferably Gram positive bacteria, preferably bacteria of the genus Bacillus, and preferably species of Bacillus thuringiensis) was not previously known. While the art taught of the existence of particular, symbiotic nematodes and certain bacteria as discussed above, the art did not teach or suggest that nematodes could be used as an excellent source for isolating very unique strains of other types of insecticidal bacteria, particularly unique strains of Bacillus thuringiensis.